Abstract/Summary: Caries and dental trauma are major oral health burdens. Globally, 21% of children (age 6 to 11 years) have caries in their permanent teeth. In the US, 18% of school children experience dental trauma. Dental pulp injury due to caries or trauma, leads to inflammation, which if left untreated, results in necrosis. Traditional therapeutics of necrotic immature permanent teeth allows for infection control, but support neither root development nor restoration of the immunocompetence of the pulp. To date, no clinical therapy exists that promotes root canal disinfection and can consistently guide the growth and development of pulp and dentin in necrotic teeth. Thus, there is a pressing need to develop a strategy for predictable pulp-dentin regeneration in a bacteria-free environment which may ultimately lead to the establishment of novel therapeutics to treat immature teeth with pulpal necrosis. The objective of this application is to develop a novel strategy to stimulate pulp and dentin regeneration by engineering an injectable collagen-fibril matrix system with heterogeneous stiffness and selected growth factors (GFs), which will first require the attainment of a bacteria-free niche. Our first hypothesis is that electrospinning can be used to develop non-toxic and antimicrobially effective 3D tubular drug delivery constructs for root canal disinfection that release initially high amount of antibiotics and sustain its effects for several days. The proposed construct will be evaluated for its release properties and cell compatibility in vitro. Antimicrobial properties will be determined using an in vitro infected tooth slice model and an in vivo model of immature dog teeth with periapical disease (Aim 1). Our second hypothesis is that dental pulp stem cell transplantation within a stiffer collagen matrix added with bone morphogenetic protein-2 (BMP-2) or within a more compliant matrix added with vascular endothelial growth factor (VEGF), when concentrically injected into a disinfected root canal, will lead to dentin and pulp regeneration, respectively. We propose to optimize the novel self-assembling collagen-fibril matrix system by evaluating the cell viability, proliferation, apoptosis and differentiation to endothelial and odontoblast cells using in vitro cell-based assays and a well- established in vivo tooth slice SCID mice model (Aim 2). Finally, the regenerative capacity of the optimal and standardized injectable collagen-fibril matrix system will be evaluated using an in vivo model of immature dog teeth with periapical disease after disinfection with the drug delivery construct (Aim 3). This application is highly innovative as we propose, for the first time, the clinical role of a cell-friendly electrospun 3D tubular drug delivery construct for root canal disinfection. Further, we propose to couple this therapy with a unique regenerative strategy using injectable and highly tunable collagen-fibril matrices to amplify dental pulp stem cell differentiation to form pulp and dentin in the appropriate locations. The proposed research is significant because it will expedite the establishment of a reliable regenerative therapeutics to treat necrotic immature permanent teeth.